CA2999332A1 - Crystalline form of 2-(2,6-dichlorophenyl)-1-[(1s,3r)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1h)-yl]ethanone for the treatment of parkinson's disease - Google Patents

Abstract

The invention provides a crystalline form of 2-(2,6-dichlorophenyl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone, and pharmaceutical compositions thereof. The invention further provides methods of using a crystalline form of 2-(2,6-dichlorophenyl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone to treat cognitive impairment associated with Parkinson's disease or schizophrenia.

Description

-CRYSTALLINE FORM OF

2-(2,6-DICHLOROPHENYL)-1-1(1S,3R)-3-(HI'DROXYMETHYL)-5-(3-HYDROXY-3-METHYLBUTYL)-1-METHYL-

3,4-DIHYDROISOQUINOLIN-2(1H)-YLIETHANONE FOR THE TREATMENT OF PARKINSON'S
DISEASE
The present invention provides a crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-543-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone, pharmaceutical compositions thereof, methods of using the same, and processes for preparing the same.
The dopaminergic pathway is associated with a number of disorders including Parkinson's disease, schizophrenia and attention deficit-hyperactivity disorder (ADHD).
Parkinson's disease is a chronic, progressive, neurodegenerative disorder characterized by the loss of dopaminergic neurons in the brain. Parkinson's disease manifests in resting tremor along with other motor syinptoms (e.g. akinesia and bradykinesia) and non-motor symptoms (e.g. cognitive impairment, sleep disorders, and depression). Current therapy for treatment of Parkinson's disease includes dopamine precursors such as levodopa, and dopamine agonists such pramipexole. Such direct acting dopamine therapies are limited in effectiveness due in part to high dose associated cognition impairment, seizure risk (as shown in rodents), and tolerance development. There remains a significant unmet need for safe and effective treatment of Parkinson's disease.
Allosterie modulators are agents that remotely alter the interactions of ligands with their receptors by modifying the ligand-binding environment. An example of this type of modulation is when the binding of a modulator to an allosteric (secondary) site produces a conformational change in the receptor protein that is transmitted to the ligands orthosteric (primary) binding site. The quality of the allosteric effect is said to be positive if the modulator facilitates or potentiates an interaction or negative if it inhibits an interaction of the ligand with the orthosteric binding site.
Compounds of the present invention are positive allosteric modulators (PAM) of the dopamine 1 receptor (D1). As such, compounds of the present invention are useful for the treatment of conditions in which D1 plays a role such as Parkinson's disease and schizophrenia, including relief of associated symptoms such as mild cognitive impairment. Compounds of the present invention are also believed to be useful in treating symptoms of Alzheimer's disease such as cognitive impairment.
Compounds of the present invention are also useful in improving motor symptoms in Parkinson's disease as a monotherapy. As well, compounds of present invention are believed useful in treating depression and attention deficit-hyperactivity disorder (ADHD).
The present invention provides a new crystalline form of the compound, 242,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone, which is a positive allosteric modulator (PAM) of the dopamine 1 receptor (D1) and, as such, is useful in treatment of the disorders discussed above. A crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone is believed to provide advantages in manufacturing and development of the compound itself as well as related compounds such as cocrystalline form of a composition comprising 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yl]ethanone and

4-hydroxybenzoic acid.
U.S. Patent No. 8962654 discloses certain 3,4-dihydroisoquinolin-2(1H)-y1 compounds as positive allosteric modulators (PAM) of the dopamine 1 receptor (D1) including 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone and a cocrystalline form of a composition comprising 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-

5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihyciroisoquinolin-2(111)-yl]ethanone and 4-hydroxybenzoic acid.
The present invention provides a crystalline form of 2-(2,6-dichloropheny0-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone.
Further, the present invention provides a crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yliethanone characterized by an X-ray powder diffraction pattern using CuKa radiation having a diffraction peak at diffraction angle 2-theta of 9.15 in combination with one or more peaks selected from the group consisting of 19.4 , 16.1 , and 16.6 ; with a tolerance for the diffraction angles of 0.2 degrees.
Further, the present invention provides a pharmaceutical composition comprising a crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(11-/)-yflethanone, and a pharmaceutically acceptable carrier, diluent or excipient.
Further, the present invention provides a method of treating Parkinson's disease comprising administrating to a patient in need thereof an effective amount of a crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(111)-yliethanone.
Further, the present invention provides a method of treating mild cognitive impairment associated with Parkinson's disease comprising administrating to a patient in need thereof an effective amount of a crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone.
Further, the present invention provides a crystalline form of 242,6-dichloropheny1)-1-[(1S,3R)-3-(1iydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone for use in therapy.
Further, the present invention provides a crystalline form of 242,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone characterized by an X-ray powder diffraction pattern using CuKa radiation having a diffraction peak at diffraction angle 2-theta of 9.15 in combination with one or more peaks selected from the group consisting of 19.4 , 16.1 , and 16.6 ; with a tolerance for the diffraction angles of 0.2 degrees for use in therapy.
Further, the present invention provides a crystalline form of 242,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone for use in the treatment of Parkinson's disease.
Further, the present invention provides the use of a crystalline form of 2-(2,6-dichloropheriy1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)- I-methyl -3,4-dihydroisoquinolin-2(1H)-yflethanone for the manufacture of a medicament for Parkinson's disease.
Compound 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(111)-yflethanone is represented by formula la.

H
OH

0 CI lel La A compound of formula la may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of formula la are useful including Parkinson's disease and schizophrenia. Such other drug(s) may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with a compound of formula Ia. When a compound of formula Ia is used contemporaneously with one or more other drugs, a pharmaceutical unit dosage form containing such other drugs in addition to the compound of formula Ia is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of formula la. Examples of other active ingredients effective in the treatment of Parkinson's disease that may be combined with a compound of formula Ia, either administered separately or in the same pharmaceutical composition include, but are not limited to:
(a) dopamine precursors such as levodopa; melevodopa, and etilevodopa; and (b) dopamine agonists including pramipexole, ropinirole, apomorphine, rotigotine, bromocriptine, cabergoline, and pergolide.
It is understood that compounds of the present invention may exist as stereoisomers. Embodiments of the present invention include all enantiomers, diastereomers, and mixtures thereof. Preferred embodiments are single diastereomers, and more preferred embodiments are single enantiomers.
As used herein, the term "cocrystal" refers to a multiple component crystalline solid form comprising two compounds where the association of compounds is primarily through non-covalent and non-ionic chemical interactions such as hydrogen bonding. In the pharmaceutical arts, a cocrystal typically comprises a first compound which is an active pharmaceutical ingredient and a second compound which is referred to as a guest compound or coformer. A cocrystal may be distinguished from a crystalline salt form in that the first compound remains essentially uncharged or neutral. A cocrystal may be distinguished from a crystalline hydrate or solvate form in that the guest compound is not exclusively water or a solvent. A preferred cocrystal is a stable form having a suitable melting point.
As used herein, the term "patient" refers to an animal such as a mammal and includes a human. A human is a preferred patient.
It is also recognized that one skilled in the art may treat Parkinson's disease by administering to a patient presently displaying symptoms an effective amount of the compound of formula Ia. Thus, the terms "treatment" and "treating" are intended to refer to all processes wherein there may be a slowing, interrupting, arresting, controlling, or stopping of the progression of an existing disorder and/or symptoms thereof, but does not necessarily indicate a total elimination of all symptoms.
It is also recognized that one skilled in the art may treat Parkinson's disease by administering to a patient at risk of future symptoms an effective amount of the compound of formula la and is intended to include prophylactic treatment of such.
As used herein, the term "effective amount" of a compound of formula Ia refers to an amount that is a dosage, which is effective in treating a disorder, such as Parkinson 's disease described herein. The attending diagnostician, as one skilled in the art, can readily determine an effective amount by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining an effective amount or dose of a compound of formula la, a number of factors are considered, including, but not limited to the compound of formula la to be administered;
the co-administration of other agents, if used; the species of mammal; its size, age, and general health; the degree of involvement or the severity of the disorder, such as Parkinson's disease; the response of the individual patient; the mode of administration;
the bioavailability characteristics of the preparation administered; the dose regimen selected;
the use of other concomitant medication; and other relevant circumstances.

-6-A compound of formula la can be administered alone or in the form of a pharmaceutical composition with pharmaceutically acceptable carriers, diluents or excipients. Such pharmaceutical compositions and processes for making the same are known in the art (See, e.g., Remington: The Science and Practice of Phaxmacy, D.B.
Troy, Editor, 21st Edition., Lippincott, Williams & Wilkins, 2006).
Human D1 Receptor Positive Allosteric Modulation Assay

-7-HEK293 cells stably expressing the human D1 receptor are generated via retroviral gene transduction using the pBABE-bleo vector and the Phoenix retroviral system. The cells are gown in DMEM/F12 (Gibco) supplemented with 10% calf serum, 20mM HEPES, 2mM glutamate, and 1504mlzeocin at 37C in 5% CO2. At approximately 80% confluency, the cells are harvested using 0.25%
trypsin/EDTA, suspended in FBS plus 8% DMSO, and stored in liquid nitrogen. On the day of the assay, cells are thawed and re-suspended in ST1M buffer (Hanks Balanced Salt Solution supplemented with 0.1% BSA, 20mM HEPES, 20011M IBMX, and 1001.1,M ascorbic acid). A test compound is serially diluted (1:3) in DMSO and then further diluted 1:40 into STIM buffer containing 2X an EC20 concentration of dopamine. An EC20 concentration of dopamine is defined as the concentration that increases cyclic AMP up to 20% of the maximum amount that can be induced by dopamine; in this assay, the EC. is 5p,M, and the EC20 is generally 12nM. Twenty-five pl of this solution is mixed with 25111 of cell suspension (1,250 cells) and dispensed into each well of 96-well, half-area plates; the fmal DMSO concentration is 1.25%. Plates are incubated at 250C for 60 min. cAMP production is quantified using HTRF detection (CisbioTM) as per vendor instructions: lysis buffer containing anti-cAMP cryptate and D2-conjugate (25111 of each) are added to the wells, plates are incubated for another 60-90 min, and fluorescence is detected using an EnVision plate reader (PerkinElmerru). Data are converted to cAMP
concentrations using a cAMP standard curve, and analyzed as an absolute EC50 using a 4-parameter nonlinear logistic equation (AbaseTM v5.3.1.22). The absolute EC50 for a positive allosteric modulator is calculated as the concentration generating a half-maximal amount of cAMP, based on a window ranging from the dopamine EC20, which defines the minimum response, to the EC. response, defined by the addition of 5 M
dopamine.
In the above assay, the compound of Reference Compound 1 exhibits an absolute EC50 ' of 4 59 + 0.96 nM (SEM; n=7) with a maximum response of 79.7 +
6.2 % at the human D1 receptor. The cocrystal of Reference Compound 2 exhibits an absolute of 1.11 -4- 0.11 nM (SEM; n=2) at the human D1 receptor. These data demonstrate the compound of Reference Compound 1 is a positive allosteric modulator of the human Dl receptor.

8 Anti-Parkinson effects of compounds of the invention can be determined using procedures well known in the art such as animal models of locomotor activity.
For example, the compound of Reference Compound 1 shows effects on basal (habituated) locomotor activity and on reserpine-induced akinesia in humanized dopamine 1 receptor (D1) knock-in mice.
Generation of Human 1)1 Receptor Knock-in Mouse A transgenic mouse in which the inurine dopamine 1 (D1) receptor is replaced by its human counterpart may be generated by standard techniques. For example, mouse genomic fragments are subcloned from the RP23 bacterial artificial chromosome library and recloned into a PGK-neo targeting vector. The mouse open reading frame is replaced with the human DI receptor open reading frame in exon 2. A neo selection marker upstream of exon 2 is flanked by frt sites for later removal. The flanking of exon 2 by loxP selection sites allows for the option to generate D1 knock-out mice by crossing with mice expressing the cre nuclease gene.
The C57BL/6 N embryonic stem cell line B6-3 is grown on a mitotically inactivated feeder layer of mouse embryonic fibroblasts in high-glucose DMEM
with 20% fetal bovine serum and 2x106 unit/1 leukemia inhibitory factor. Ten million embryonic stem cells plus 30 micrograms of linearized vector DNA are electroporated and subjected to 0418 selection (200 micrograms/ml). Clones are isolated and analyzed by Southern blotting.
A clone containing the expected size insert is inserted into blastocysts and the resulting mice are genotyped by PCR. A male chimera is crossed with a female containing the Flp nuclease gene to eliminate the selection marker. Progeny containing the human D1 receptor without the selection marker are identified by PCR. A
male heterozygote is mated with female C57BL/6 mice. Male and female progeny containing the human D1 receptor are mated and homozygotes are identified by PCR.
Behavior and reproduction of the homozygotes is normal, and the colony is maintained in the homozygote state for succeeding generations.
Basal (habituated) Locomotor Activity Locomotor activity is measured using an automated system to track movement in mice. Human D1 receptor knock-in mice are placed in chambers and allowed to

-9-habituate to the chambers for 60 mins. During this time, they show reduced locomotion over time. Following administration of a compound of the invention, animal movement is increased in a dose-dependent fashion.
More specifically, locomotor activity boxes are situated in rectangular frames with infrared beams for measurement of motor activity (horizontal and vertical activity) called ambulations. Locomotor activity is recorded between time of 7:30 and 15:00 hours.
Mice are randomly assigned to treatment groups as shown in Table 1. Each mouse is placed individually into one of the locomotor activity boxes for 60 minutes habituation. Mice are then dosed orally and total number of ambulations is recorded per

10 minutes for each mouse over a 60 minutes period. In mice with reserpine pretreatment, no previous habituation period is included. Thus, immediately after dosing, the total number of ambulations is measured for 60 minutes. Data is transferred from the software/computers to spreadsheets for further analysis. Statistical analysis is carried out using one-way ANOVA followed by post-hoc analysis using Fishers' LSD or Dunnett's test.
In the basal (habituated) locomotor assay, Reference Compound 1 facilitates movement in mice in a dose responsive manner (Table 1). This demonstrates Reference Compound 1 is effective in locomotor activation of animals that are habituated to the environment.

Table 1 Basal Locomotor Activity (Total Ambulations for 60 Test Compound min.) (Means, SEM, %SE) Means 542 vehicle SEM 111 %SE 30 Reference Compound 1 Means 542 (1 mg/kg) %SE 10 Reference Compound 1 Means 1118 *

(3 mg/kg) %SE 26 Reference Compound 1 Means 1818 ** @

(6 mg/kg) %SE 22 Reference Compound 1 Means 3047 *** @

(10 mg/kg) %SE 10 Reference Compound 1 Means 4623 *** @

(30 mg/kg) %SE 11 **p<0.01, ***p<0.001 compared to vehicle (unpaired t-test) @ p<0.0I compared to vehicle One-way ANOVA Dunnett's Multiple Comparison Test Reversal of Reserpine-Induced Akinesia Reserpine is a catecholamine depleting agent (depletes dopamine and noradrenalin) and 18-24 hours after treatment mice become akinetic and have reduced locomotor activity counts. Reserpine-induced akinesia is assessed by measuring the effect of compounds on locomotor activity approximately! 8-24 hours after a single dose of 0.15 or 0.3 mg/kg reserpine subcutaneous. The equipment used is the same as that used for habituated locomotor activity as previously discussed.
Male humanized dopamine DI receptor knock-in mice are randomly assigned to treatment groups as shown in Table 2. Each mouse is placed individually into one of the locomotor activity boxes. Ambulations per 10 minutes for each mouse is measured for up

-11-to 60 mins after dosing. Thus, effects on reserpine-induced exploratory behavior is assessed for a total of 60 minutes. Data is transferred from the software/computers to spreadsheets for further analysis. Statistical analysis is carried out using one-way ANOVA followed by post-hoc analysis using t-test.
In the above assay, the compound of Reference Compound 1 reverses the effects of reserpine treatment and restores movement in mice in a dose responsive manner (Table 2). This demonstrates that the compound of Reference Compound 1 is effective in an in vivo model of Parkinson's disease.

-12-Table 2 Locomotor Activity (Total Ambulations for 60 Test Compound min.) (Means, SEM, %SE) Vehicle Control Means 1629 (no reserpine) %SE 12 Vehicle + Reserpine Means 1336 (0.15 mg/kg) %SE 14 Vehicle + Reserpine Means 640 (0.3 mg/kg) %SE 9 Reference Compound 1 Means 4623 ***
(10 mg/kg) + Reserpine SEM 486 (0.15 mg/kg) %SE 11 Reference Compound 1 Means 6222 ***
(30 mg/kg) + Reserpine SEM 659 (0.15 mg/kg) %SE 11 Reference Compound 1 Means 4056 ***
(30 mg/kg) + Reserpine SEM 548 (0.3 mg/kg) %SE 13 *p<0.05, **p<0.01, ***p<0.001 compared to vehicle (unpaired t-test) @ p<0.01 compared to vehicle One-way ANOVA Dunnett's Multiple Comparison Test Compounds of formula la may be prepared by processes known in the chemical art or by a novel process described herein. A process for the preparation of a compound of formula Ia and novel intermediates for the manufacture of a compound of formula Ia, provide further features of the invention and are illustrated by the following procedures.
Generally, a compound of formula Ia may be prepared from a compound of formula H where Pgl represents a suitable hydroxyl protecting group (Scheme 1).

-13-Particular values of Pgl include tert-butyl(dimethypsily1 and tert-butyl(diphenyl)silyl.
More specifically, a compound of formula II where Pgl is tert-butyl(dimethyl)sily1 is reacted with tetrabutylanunonium fluoride in a solvent such as tetrahydrofitran to provide a compound of formula Ia. A compound of formula II where Pgl is tert-butyl(dimethyl)sily1 may be prepared by reacting a compound of formula III with a methyl lithium in a suitable solvent. Suitable solvents include tetrahydrofuran. A
compound of formula III where Pgl is tert-butyl(dimethypsily1 may be prepared by reacting a compound of formula IV with hydrogen in the presence of a suitable transition metal catalyst such as 1,1'-bis(di-i-propylphosphino)ferrocene(1,5-cyclooctadiene)rhodium(1) tetrafluoroborate. The reaction is conveniently carried out in a solvent such as methanol. A compound of formula IV where Pgl is tert-butyl(dimethyl)sily1 may be prepared by reacting a compound of formula V
with ethyl acrylate in the presence of a transition metal coupling catalyst such as palladium acetate, a ligand such as tri-o-tolylphosphine and a base such as triethyamine. The reaction is conveniently carried out in a solvent such as acetonitrile. A
compound of formula V may be prepared by acylating a compound of formula VIII with 2,6-dichlorophenylacetic acid in the presence of an activating agent such as benzotriazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate and a base such as triethylamine.
The reaction is conveniently carried out in a solvent such as ditnethyl form amide.

-14-Scheme I
OH OH
0"Pg1 OH
.õ1 41i ; CI deprotection õ.
41I N;*s CI
0 I *

II la MeLi TIE

0-Pgi catalyst 0-Pg1 H2, Me011 õ1 ,õ
Ott ; 010 N.. CI

ci 0CI 00 III Iv A
ethyl acrylate catalyst Et3N, CH3CN
Br O'Pg1 Br 0-Pg1 2,6-diClphenylacetic acid ..= =activating reagent, DMF c, NH
o Ci VI II V
Alternatively, a compound of formula 11 may be prepared from a compound of formula VI where Pgl is a suitable hydroxyl protecting group (Scheme 2). More specifically, a compound of formula VI where PgI is tert-butyl(dirnethypsily1 is acylated with 2,6-dichlorophenylacetic acid in the presence of an activating agent such as 1,1'-carbonyldiimidazole to provide a compound of formula II. The reaction is conveniently carried out in a solvent such as tetrahydrofuran. A compound of formula VI

-15-may be prepared by reducing a compound of formula VII with hydrogen in the presence of a catalyst such as palladium. The reaction is conveniently carried out in a solvent such as ethanol. A compound of formula VII may be prepared by coupling a compound of formula VIII with 2-methylbut-3-en-2-ol in the presence of a catalyst such as palladium acetate, a ligand such as 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl and a base such potassium carbonate. The reaction is conveniently carried out in a solvent such as dimethylformamide. A compound of formula VIII made be prepared in a multistep fashion from a compound of formula X through an intermediate compound of formula TX
as described in the Preparations and Examples. A compound of formula X may be prepared as described in the Preparations and Examples.
Scheme 2 OH OH
o-Pgi 5% Pd/C Pgi Et0H I 2,6-diClphenylacetic acid 1,1'-carbonyldiimidazole, THF
NH SI NH
VII VI
2-inethylbut-2-en-3-ol catalyst DMF
Pgi Br Br Br 0 õI
OH
00 NH op NH miti NH
VIII ix X
As used herein, "DMSO" refers to dimethylsulfoxide; "Tris" refers to trishydrox ymethylaminomethane; "DTT" refers to dithiothreitol; "HEC" refers to hydroxycthyl cellulose; "DMEM" refers to Dulbecco's Modified Eagle's Medium;
"MS"

-16-refers to mass spectrum; and "1-IF,PS" refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
Preparation 1 Synthesis of methyl 2-bromo-D-phenylalaninate hydrochloride.
Br 0 4110 = ''"

HCI
Dissolve 2-bromo-D-phenylalanine (22.4 g, 91.8 mmol) in methanol (459 mL).
Add acetyl chloride (65.3 mL, 917.7 mmol) at room temperature. Stir for 36 hours.
Concentrate under reduced pressure to give the title compound (27.2 g, 92.3 mmol). MS
(nth): 258 (M+1).
Alternative synthesis of methyl 2-bromo-D-phenyialaninate hydrochloride.
Add acetyl chloride (562.79 g, 7.17 mol) to methanol (10.00 L) at 0 C in an appropriate vessel. Heat the mixture to 17.5 C and stir. After 30 minutes add 2-bromo-D-phenylalanine (500.00 g, 2.05 moles) and heat to reflux. After 4 hours, cool to 20 C
and remove the solvent under reduced pressure to give the title compound (589 g, 1.96 mol) as an off-white solid. MS (m/z): 258(M-C1 (79Br)), 260(M-C1(81Br)).
Preparation 2 Synthesis of methyl 2-bromo-N-(nethoxycarbony1)-D-phenylalaninate.
Br .10 0 Y

Dissolve methyl 2-bromo-D-phenylalaninate hydrochloride (27.2 g, 92.3 mmol) in dichloromethane (923 mL) and water (185 mL). Add sodium bicarbonate (31.0 g, 369.4 mmol) and methyl chloroformate (7.86 mL, 101.6 mmol) at room temperature. Stir the mixture 2.5 hours. Dilute with water and extract with dichloromethane. Dry the dichloromethane extracts over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel chromatography eluting with ethyl acetate:

-17-hexanes (10-75% gradient) to give the title compound (29.1 g, 92.1 mmol). MS
(m/z):
316 (M+1).
Alternative synthesis of methyl 2-bromo-N-(methoxycarbony1)-D-phenylalaninate.
Add water (2.94 L) and sodium hydrogen carbonate (648.25 g, 7.64 mol) to methyl 2-bromo-D-phenylalaninate hydrochloride (580 g, 1.91 mol) in dichloromethane (9.86 L) at 10 C in an appropriate vessel. After 5 minutes add methyl chloroformate (198.53 g, 2.10 mol) and stir the mixture at 20 C. After 3 hours add water (2.5 L) and separate the layers. Extract the aqueous with dichloromethane, dry the combined organic extracts over sodium sulfate and concentrate under reduced pressure to give the title compound (556 g, 1.74 mol). MS (m/z): 315.8(M+1 ("Br)), 317.8(M+1 (8IBr)).
Preparation 3 Synthesis of dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate.
Br 0 Y

Stir a mixture of methyl 2-bromo-N-(methoxycarbony1)-D-phenylalaninate (29.1 g, 92.10 mmol) and paxaformaldehyde (9.13 g, 101.3 mmol) in glacial acetic acid (115 mL, 2.01 mol) and concentrated sulfuric acid (38.4 mL, 719.9 mmol) at room temperature for 7 hours. Partition between water and ethyl acetate. Separate the layers and extract the aqueous layer with ethyl acetate. Combine the ethyl acetate extracts and dry over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel chromatography eluting with ethyl acetate: hexanes (5-40% gradient) to give the title compound (27.6 g, 84.0 mmol). MS (m/z): 328 (M+1).
Preparation 3a Synthesis of dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate and (3R)-5-bromo-2-methoxycarbony1-3,4-dihydro-1H-isoquinoline-3-carboxylic acid.

-18-Br 0 Br 0 CY- OH

Y Y

To acetic acid (4.29 L) at 10 C in an appropriate vessel, add 2-bromo-N-(methoxycarbony1)-D-phenylalaninate (572 g, 1.81 mol) and paraformaldehyde (205.86 g, 2.17 mol). After 10 minutes slowly add concentrated sulfuric acid (2.63 kg, 26.83 mol) and then stir at 35 C. After 12 hours, cool to 15 C and add water (7.5 L) and ethyl acetate (6 L). Separate the layers and re-extract the aqueous with ethyl acetate (2.5 L).
Dry the combined organic extracts over sodium sulfate, filter and concentrate under reduced pressure to give a mixture of the title compounds with acetic acid (640 g, 1.69 moles). Mass spectrum (m/z): 3a: 327.95(M+1 ("Br)), 330.05(M+1 (81Br)). 3b:
314(M+1 (79Br)), 315.95(M+1 (81Br)).
Preparation 4 Br0 40 0, NH
HCI
Synthesis of methyl (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride.
Dissolve dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate (27.55 g, 84.0 mmol) in 5N hydrochloric acid (330.6 mL, 1.65 mol) and heat to reflux for three days. Concentrate under reduced pressure to give a white solid. Wash the solid with diethyl ether and dry under vacuum at 40 C overnight to give (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride (1:1) (20.8 g, 71.1 mmol).
Add acetyl chloride (50.6 mL, 711.0 mmol) to a 0 C mixture of (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride (1:1) (20.8 g, 71.1 mmol) in methanol (474 mL). Warm to room temperature and stir for 36 hours. Concentrate under reduced pressure and dry to give the title compound (21.9 g, 71.4 mmol). MS
(m/z): 270 (M+1).

-19-Preparation 5 Synthesis of (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride.
Br 0 HC
H
Add water (1.3 L) and 36.5% hydrochloric acid (9.07 Kg, 90.81 moles) to a mixture of dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate and (3R)-5-bromo-2-methoxycarbony1-3,4-dihydro-1H-isoquinoline-3-carboxylic acid (Preparation 3a) (520 g, 1.27 moles) in an appropriate vessel and stir the mixture at 95 C.
After 12 hours cool the mixture to 10 C and stir for 15 minutes. Filter the mixture and dry the solid under vacuum at 40 C give the title compound (332 g, 1.13 moles). MS
(m/z): 256.1(M-C1 ("BO), 258(M-C1(81Br)).
Preparation 6 Synthesis of 2-tert-buty1-3-methyl-(3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate.
Br 0 14.
".

Dissolve methyl (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylate hydrochloride (21.0 g, 68.5 mmol) in 1,4-dioxane (685 mL). Add saturated sodium bicarbonate solution (685 mL, 17.5 mol) and di-tert-butyldicarbonate (29.9 g, 137.0 mmol) at room temperature. Stir the biphasic mixture for 90 min. Extract with ethyl acetate. Dry the ethyl acetate over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel chromatography eluting with ethyl acetate:
hexanes (5-50% gradient) to give the title compound (19.5 g, 52.7 mmol). MS
(m/z): 270 (M4B0C+1).

-20-Preparation 7 Synthesis of [(3R)-5-bromo-1,2,3,4-tctrahydroisoquinolin-3-yl]methanol.
Br OH
*NH
Add lithium aluminium hydride (2 L, 2.00 mol, 1M in THF) to (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride (325.4 g, 1.11 mol) in tetrahydrofuran (4.88 L) at -35 'V in an appropriate vessel, then warm to 25 C over 60 minutes and stir. After 3 hours, cool the mixture to -5 C then add water (76 mL), 15%
w/w aqueous sodium hydroxide (76 mL) and water (228 mL). Heat the mixture to 25 C, add anhydrous magnesium sulfate (750 g) and stir. Filter the mixture and concentrate under reduced pressure to give a solid. Add dichloromethane (690 mL) to the solid and slurry for 30 minutes before filtration to give a solid. Dry the solid under vacuum at 35 C to give the title compound (148.9 g, 0.55 mol). MS (nth): 242(M+1 ("Br)), 244(M+1 (81Br)).
Preparation 8 Synthesis of tert-butyl (3R)-5-bromo-3-(hydroxymethyl)-3.4-dihydro-1H-isoquinoline-2-carboxylate.
Br OH
NyOl<

Add methanol (10.1 mL, 248.5 mmol) and lithium borohydride (99.4 mL, 198.8 mmol, 2 M in THF) to a solution of 2-tert-buty1-3-methyl-(3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate (18.4 g, 49.7 mmol) in tetrahydrofuran (497 mL) at room temperature on a water bath. Stir 40 min and quench the reaction with water.
Extract with ethyl acetate. Dry the ethyl acetate extracts over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel chromatography eluting with ethyl acetate: hexanes (5-80% gradient). Dry under high vacuum overnight

-21-to give the title compound as a white solid (19.1 g. 55.8 mmol). MS (m/z): 286 (M-tBu+1).
Preparation 9 Synthesis of (3R)-5-bromo-3-({ [tert-butyl(dimethyl)silyl]oxy}methyl)-1,2,3,4-tetrahydroisoquinoline.
I j<
Si Br= 0' '`=
N H
Add trifluoroazetic acid (75.5 mL, 998.3 mmol) to solution of tert-butyl (3R)-bromo-3-(hydroxymethyl)-3,4-dihydro-1H-isoquitioline-2-carboxylate (15.5 g, 45.3 mmol) in dichloromethane (226 mL) at room temperature. Stir 30 min and concentrate under reduced pressure. Dry under vacuum to give [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]nethanol; 2,2,2-trifluoroacetic acid as a wet solid. Dissolve [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methanol; 2,2,2-trifluoroacetic acid in dichloromethane (753 mL). Add 1H-imidazole (51.3 g, 753 mmol), N,N-Dimethy1-4-pyridinamine (460 mg, 3.77 mmol), and t-butyldimethylchlorosilane (13.6 g, 90.4 mmol).
Stir at room temperature overnight. Add saturated ammonium chloride solution and extract with dichloromethane. Dry the dichloromethane extracts over sodium sulfate, filter, and concentrate under reduced pressure. Combine with the crude product from a substantially same reaction run with 19.4 mmol of tert-butyl (3R)-5-bromo-3-(hydroxymethyl)-3,4-dihydro-1H-isoquinoline-2-carboxylate. Purify the residue by silica gel chromatography eluting with ethyl acetate: hexanes (5-40% gradient) to give the title compound (14.3 g, 40.1 mmol). MS (m/z): 356 (M+1).
Alternative synthesis of (3R)-5-bromo-3-({ [tert-butyl(dimethyl)silyl]oxy) methyl)-1,2,3,4-tetrahydroisoquinoline.
Add tert-butyldimethylchlorosilane (193.7 g, 1.29 mol) to a mixture of [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]nethanol (148.9 g, 0.58 mol), 1H-imidazole

-22-(202.9 g, 2.92 mol), 4-dimethylatninopyridine (0.72 g, 5.84 rnmol) and N,N-dimethylformamide (1.04 L) in dichlormethane (2.61 L) at 20 C and stir in an appropriate vessel. After 3 hours, cool the mixture to 10 C and add saturated aqueous ammonium chloride solution (1.3 L). Extract the aqueous with dichloromethane and wash the combined organic extracts with brine (2 x 2L), dry over anhydrous sodium sulfate and concentrate under reduced pressure to give a residue. Dissolve the residue in methyl tert-butyl ether (1.5 L) and wash with brine (2 x 1 L). Dilute the organic phase with toluene (5 L) and concentrate under reduced pressure to give a residue.
Add toluene (2.6 L) to the residue and concentrate under reduced pressure to give the title compound (210 g, 0.53 mol). MS (m/z). 356(M+1 ("Br)), 358(M+1 (81Br)).
Preparation 10 Synthesis of (3R)-5-bromo-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3,4 dihydroisoquinolirte.
I ,<"
Br 0' Dissolve (3R)-5-bromo-3-(fitert-butyl(dimethypsilyl]oxy}methyl)-1,2,3,4-tetrahydroisoquinoline (4.2 g, 11.8 mmol) in diethyl ether (118 mL). Add N-chlorosuccinimide (2.36 g, 17.7 mmol). Stir 30 min at room temperature and concentrate under reduced pressure. Dissolve the residue in potassium hydroxide (42.0 mL, 30.3 mmol, 5% in Me0H) and stir for 30 min at room temperature. Pour into water and extract with dichloromethane. Dry the dichloromethane extracts over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel chromatography eluting with ethyl acetate: hexanes (5-100% gradient) to give the title compound (3.40 g, 9.59 mmol). MS (tn/z): 354 (M+1).
Alternative synthesis of (3R)-5 -bromo-3-( [tert-butyl(dimethypsilyl]oxy}methyl)-3,4 dihydroisoquinoline.

-23-Add N-chlorosuccinimide (106.7 g, 0.79 mol) to a solution of (3R)-5-bromo-3-(f[tert-butyl(dimethypsilyfloxy}methyl)-1,2,3,4-tetrahydroisoquinoline (220 g, 0.52 mol) in tetrahydrofuran (3.85 L) at 20 C in an appropriate vessel and stir. After 30 minutes concentrate the mixture under reduced pressure and dissolve the residues in 5%
w/w potassium hydroxide in methanol (2.2 L, 1.69 moles) and stir at 20 C. After 30 minutes, add the mixture to water (3 L) and extract three times with dichloromethane (3 xl L).
Dry the combined organic extracts over anhydrous magnesium sulfate and concentrate under reduced pressure to give the title compound (210 g, 0.50 mol). MS (m/z):

354(M+1 ("Br)), 356(M+1 ("Br)).
Preparation 11 Synthesis of (1S,3R)-5-bromo-3-( fitert-butyl(dimethypsilyl]oxylmethyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline.
I
si-Br Dissolve (3R)-5-bromo-3-(fitert-butyl(dimethyl)silyl]oxy}methyl)-3,4 dihydroisoquinoline (3.4 g, 9.59 mmol) in diethyl ether (160 mL). Cool to -78 C on a dry ice ¨ acetone bath. Add methylmagnesium chloride (26.9 mL, 80.6 mmol, 3M
in THF) dropwise. Warm the reaction mixture slowly to room temperature and stir overnight. Quench with saturated ammonium chloride solution slowly. Extract with dichloromethane and dry over sodium sulfate, filter, and concentrate under reduced pressure. Combine with the crude product from a substantially same reaction run with 1.73 mmol of (3R)-5-bromo-3-({[tert-butyl(dimethypsilyfloxy}methyl)-3,4 dihydroisoquinoline. Purify the combined residues by silica gel chromatography eluting with ethyl acetate: hexanes (5-65% gradient) to give the title compound (3.78 g, 10.2 mmol): MS (m/z): 370 (M+1).
The relative configuration of compound (1S,3R)-5-bromo-3-({[tert-butyl(dimethyl)silyl]oxylmethyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline is determined

-24-by NMR spectroscopy using one-dimensional NOE experiments (1D-NOESY).
Selective excitation of the methyl group at 1.30 ppm gives rise to a NOE for Ha at 3.11 ppm. This NOE enhancement is only consistent with a configuration in which the methyl and Ha are on the same side of the ring (trans isomer) because in the cis isomer the methyl protons are too far away from Ha to show an NOE. Since the absolute chemistry for position 3 is known to be R, then the absolute chemistry at position 1 is deduced to be S.
\ ________________________________________ Br 401 Ha NH
Hc He. me Alternative synthesis of (1 S,3R)-5-bromo-34 [tert-butyl (dimethypsil yl] oxy methyl)-1 -methyl- 1,2,3,4-tetrahydroisoquino line.
1 k =
Si Br 0"
..õ
N H
Add methylmagnesium chloride (0.66 L, 1.99 mol, 3M in THF) to a solution of (3R)-5-bromo-3-({ [tert-butyl(dimethypsilyl]oxy}methyl)-3,4 dihydroisoquinoline (93.5 g, 0.24 mol) in diethyl ether (2.8 L) at -65 C in an appropriate vessel. Then heat the reaction mixture to 20 C over 2 hours and stir. After 16 hours, cool the mixture to 0 C
and quench the reaction with saturated aqueous ammonium chloride solution (2.5 L) and extract with ethyl acetate (2.5 L) and filter the mixture. Wash the combined organic extracts with brine (1 L), dry over anhydrous magnesium sulfate and concentrate under reduced pressure to give the crude title compound as an oil. Combine the oil with crude products from substantially same reactions run with 48 mmol and 229 mmol of [(3R)-5-bromo-3,4-dihydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane and purify them by silica gel chromatography eluting with ethyl acetate in hexanes (gradient 5-65% ethyl

-25-acetate) to give the title compound (151 g, 0.41 mol). MS (m/z): 370.1 (M+1 (79Br)), 372.1(M+1 (8IBr)).
Preparation 12 Synthesis of 14(1S,3R)-5-bromo-3-(fitert-butyl(dimethyl)silyl]oxy}methyl)-1-methyl-3,4-dihydroisoquinolin-2(11-/)-y1]-2-(2,6-dichlorophenypethanone.
I ks, Br 0' "=-j N CI
0CI I*
Add benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (7.97 g, 15.3 mmol) to a mixture of (1S,3R)-5-bromo-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline (3.78 g, 10.2 mmol) and 2,6-dichlorophenylacetic acid (2.30g. 11.2 mmol) in dimethylformamide (51.0 mL). Add triethylamine (2.13 mL, 15.3 mmol) and stir at room temperature hours. Dilute with water and extract with dichloromethane. Dry the dichloromethane extracts over sodium sulfate, filter and concentrate under reduced pressure.
Purify the residue by silica gel chromatography eluting with ethyl acetate: hexanes (5-50% gradient) to give the title compound (4.70 g, 8.43 mmol). MS (m/z: 556 (M+1).
Preparation 13 Synthesis of ethyl (2E)-3-{(1S,3R)-3-(fitert-butyl(dimethypsilyfloxy}methyl)-2-[(2,6-dichlorophenyl)acetyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1}prop-2-enoate.

k si 0CI el

-26-Bubble nitrogen through acetonitrile. Place tri-o-tolylphosphine (72.3 mg, 0.23 mmol), palladium (II) acetate (11.8 mg, 0.052 mmol) and acetonitrile (0.96 mL) in a microwave vessel. Stir 10 min. Add ethyl acrylate ( 0.31 mL, 2.88 mmol). Add 1-[(1S,3R)-5-bromo-3-({[tert-butyl(dimethyl)silyfloxy}methyl)-1-methyl-3,4-dihydroisoquinolin-2(1.H)-y1]-2-(2,6-dichlorophenypethanone (0.54 g, 0.96 mmol). Add triethylamine (0.40 mL, 2.88 mmol) and stir vigorously. Blow nitrogen across the surface of the reaction. Seal the vessel and heat to 160 C for 35 mm i in a microwave. Cool to room temperature and dilute with ethyl acetate. Filter the precipitate and wash with ethyl acetate. Concentrate the filtrate under reduced pressure to give a brown oil.
Combine with the crude product from a substantially same reaction run with 0.27 mmol of 1-[(1S,3R)-5-bromo-3-({[tert-butyl(dimethypsilyl]oxy}methyl)-1-methyl-3,4-dihydroisoquinolin-2(1.H)-y1]-2-(2,6-dichlorophenypethanone. Purify the combined residues by silica gel chromatography, eluting with ethyl acetate: hexanes (gradient, 0-10%) to give the title compound (0.54 g, 0.93 nunol). MS (m/z): 576 (M+1).
Preparation 14 Synthesis of ethyl 3-1(1S,3R)-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[(2,6-dichlorophenypacetyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1}propanoate.

s, In a dry box to a 85 ml Parr autoclave with stir bar and glass liner, add 1,1'-bis(di-i-propylphosphino)ferrocene(1,5-cyclooctadiene)rhodium(1) tetrafluoroborate (7 mg, 0.010 mmol). Add ethyl (2E)-3-1(1S,3R)-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[(2,6-dichlorophenypacetyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-yllprop-2-enoate (133 mg, 0.23 mmol) as a solution in anhydrous methanol (5 mL). Seal the autoclave and remove from the dry box. Purge the vessel with hydrogen and pressurize to 690 kPa of

-27-hydrogen. Stir at room temperature overnight. Vent and open the vessel.
Concentrate the reaction mixture under reduced pressure. Purify the residue by silica gel chromatography, eluting with 25% methyl t-butyl ether: hexanes to give the title compound as a clear colorless oil (121 mg, 0.21 nunol). MS (m/z): 578 (M+1).
Preparation 15 Synthesis of (1S,3R)-5-bromo-3-({[tert-butyl(dimethypsilyl]oxy) meth yl )- 1 -meth yl-1,2,3,4-tetrahydroisoquinoline hydrochloride.
Si Br 0' I `'s=
N H
HCI
Add hydrogen chloride (267.24 g, 1.02 mol, 4M in 1,4-dioxane) to (1S,3R)-5-bromo-3-({[tert-butykdimethypsilyfloxy}methyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline (419 g, 1.02 mol) in isopropyl acetate (4.19 L) at 10 C and stir for minutes in an appropriate vessel. Filter the mixture and wash the filter cake with isopropyl acetate (2.5 L), dry on the filter for 30 minutes, then under vacuum in an oven at 40 C for 16 hours to give the title compound (380 g, 0.89 mol). MS (m/z):
370(M-C1 15 (79Br)), 372(M-C1(81Br)).
Preparation 16 Synthesis of (E)-4-[(1S,3R)-3-Rtert-butyl(dimethypsi lylioxymethy1]-1 -methyl-1 .2,3.4-tetrahydroisoquinolin-5-y1]-2-methyl-but-3-en-2-ol.
HO

N H
Add (1 S,3R)-5-bromo-3-( [tert-butyl(dimethyl)silyl]oxy} meth y1)-1-methyl -1,2,3,4-tetrahydroisoquinoline hydrochloride (310 g, 723.83 nunol) and 2-methylbut-3-en-2-ol (508.95 g, 5.79 mol) to N,N-dimethylformamide (1.08 L) in an appropriate vessel

-28-and degas by bubbling nitrogen through the solution for 10 minutes. Add potassium carbonate (315.12 g, 2.28 mol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (15.32 g, 36.19 mmol) and palladium (II) acetate (8.29 g, 36.19 mmol) and degas by bubbling nitrogen through the mixture for 15 minutes then heat to 125 C. After 16 hours, cool the mixture to 20 C and dilute with ethyl acetate (1.5L) and water (2.5 L). Wash the ethyl acetate layer with brine (2.5 L), then dry over sodium sulfate and concentrate under reduced pressure to give a residue. Purify the residue by flash chromatography, eluting with 0-50% ethyl acetate in isohexanes to give the title compound (193 g, 459.1 mmol).
MS (m/z): 376(M+1).
Preparation 17 Synthesis of 4-[(1S,3R)-3-Rtert-butyl(dimethypsilylloxyrnethyl] -1-methy1-1,2,3,4-tetrahydroisoquinolin-5-y1]-2-methyl-butan-2-ol.
HO
Si =
I
N H
Add a solution of (E)-4-[(1S,3R)-3-[Etert-butyl(dimethyl)silyl]oxymethyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1]-2-methyl-but-3-en-2-ol (68 g, 168.83 mmol) in ethanol (816 mL) to a pressure hydrogenation vessel and add 5% palladium on activated carbon (35.83 g, 16.84 mmol). Purge the vessel with hydrogen gas, pressurise to 470 kPa of hydrogen gas and stir at 25 C. After 16 hours, vent the vessel and filter the reaction mixture through diatomaceous earth. Wash the diatomaceous earth with ethyl acetate and concentrate the filtrate under reduced pressure to give an oil.
Dissolve the oil in ethyl acetate (1 L) and concentrate under reduced pressure to give the title compound (64 g, 161 mmol). MS (m/z): 378.2 (M+1).

-29-Preparation 18 Synthesis of 1-[(1S,3R)-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-y1]-2-(2,6-dichlorophenyl)ethanone.
OH
s, CI

Dissolve 4-[(1S,3R)-3-Rtert-butyl(dimethypsilyl]oxymethyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1]-2-methyl-butan-2-ol (0.21 g, 0.36 mmol) in THF
(2.0 mL).
Cool on a dry ice ¨ acetone bath. Add methyl lithium (0.67 mL, 1.07 mmol, 1.6 M in diethyl ether) slowly and stir on the dry ice-acetone bath for 4 hours. Add saturated ammonium chloride solution (2 mL). Remove the dry ice-acetone bath and allow the mixture to warm to room temperature. Extract with ethyl acetate. Combine the ethyl acetate extracts; wash with brine, dry over sodium sulfate, filter, and concentrate under reduced pressure to give a clear colorless oil. Purify the residue by silica gel chromatography, eluting with ethyl acetate: hexanes (gradient, 0-30%) to give the title compound as a clear, colorless oil (0.16 g, 0.29 mmol). MS (m/z): 564 (M+1).
Alternative synthesis of 1-[(1S,3R)-3-({[tert-butyl(dimethypsilyl]oxy}methyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(110-y1]-2-(2,6-dichlorophenypethanone.
Add 1,1'-carbonyldiimidazole (112.9 g, 682.36 mmol) to a mixture of 2,6-dichlorophenylacetic acid (173.09 g, 818.83 mmol) in tetrahydrofuran (1.63 L) in an appropriate vessel and stir at 25 C. After 1 hour add a solution of 4-[(1S,3R)-3-Htert-butyl(dimethypsilyfloxymethyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1]-2-methyl-butan-2-ol (217 g, 545.89 mmol) in tetrahydrofuran (1.63 L) to the mixture, heat the mixture to 45 C and stir. After 24 hours, cool the mixture to 20 C, remove 2 L of tetrahydrofuran by concentrating under reduced pressure and dilute the residues with

-30-ethyl acetate (2.5 1.). Wash the ethyl acetate solution with saturated aqueous ammonium chloride (1.5 L), 1M aqueous sodium hydroxide (1 L), water (1 L) and brine (1.5 L). Dry the organics over anhydrous sodium sulfate and concentrate under reduced pressure to give the title compound (376 g, 532.70 mmol). MS (m/z): 564.2(M+1 (35C1)), 566.2 (M+1 (37C1)).
Reference Compound 1 Synthesis of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-ylieihanone.
OH

CI

Dissolve 1-[(1S,3R)-3-({[tert-butyl(dimethypsilyl]oxylmethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-y1]-2-(2,6-dichlorophenypethanone (0.16 g, 0.28 mmol) in THF (2.8 mL). Add tetrabutylammonitun fluoride (0.30 mL, 0.30 mmol, 1M in THF). Stir 40 min. Add saturated ammonium chloride solution and extract with ethyl acetate. Combine the ethyl acetate extracts; wash with water and brine, dry over sodium sulfate, filter, and concentrate under reduced pressure to give a residue.
Purify the residue by silica gel chromatography, eluting with ethyl acetate:
hexanes (gradient, 0-60%) to give the title compound as a white foam (0.12 g, 0.26 mmol). MS
(m/z): 450 (M+1).
Alternative synthesis of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone.
Add tetra-n-butylanunonium fluoride (651.71 mL, 651.71 nunol, 1M in THF) to a solution of 1-[(1S,3R)-3-Rtert-butyl(dimethyl)silyl]oxymethyl]-5-(3-hydroxy-3-methyl-buty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-y1]-2-(2,6-dichlorophenypethanone (400 g, 566.71 mmol) in tetrahydrofuran (4 L) at 5 *C in an appropriate vessel.
Heat the mixture to 20 C and stir. After 3 hours, remove 3 L of tetrahydrofttran by concentrating

-31-under reduced pressure and dilute the residues with ethyl acetate (2.5 L).
Wash the organics with saturated aqueous ammonium chloride (2 L), water (2 L) and brine (2 x 2 L). Dry the ethyl acetate solution over anhydrous sodium sulfate and concentrate under reduced pressure to give an oil. Dissolve this oil in 2-propanol (2.5 L) and concentrate under reduced pressure to give an oil. Purify by chiral SFC using AS-H column (50 x 250mm, 5 micron particle size) eluting with 80 % supercritical carbon dioxide and 20%
of a 0.2% solution of diethylmethylamine in isopropyl alcohol at 280 g/min. to give the title compound (182.8 g, 389.62 mmol). MS (m/z): 450.2(M+1 ("CI)), 452.2 (M+1 ("CD). Optical rotation: [a] 20D -39.4 (c = 0.95, Me0H).
Reference Compound 2 Cocrystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yl]ethanone and 4-hydroxybenzoic acid.
OH
OH

N
0 CI 1411) 0 OH
Compounds 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-mcthylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-Aethanone (402.56 mg) and 4-hydroxybenzoic acid (144.7 mg) are placed in a 40 mL vial along with a stirbar. The vial is filled to the brim with water (39 mL). The sample is stirred at 1200 rpm at 50 C
(stirplate setting). Silicon oil is dripped around the base of the vial to ensure good thermal transfer with the hotplate. A thick white slurry results with chunks of off-white solid. After an hour of slurrying, a thermometer inserted through the septum of the vial read 40.5 C, and the sample had turned into a homogenous slurry of bright white solid.
After overnight slurry, the sample is a homogenous slurry of flocculent white solid. The thermometer reads 43.1 C. Polarized light microscopy shows full birefringence.
The bright white solid is isolated by vacuum filtration and dried in place under air stream for

-32-minutes. The sample is placed in the 75 C vacuum oven for two hours to provide the title composition as a white crystalline solid (484 mg, 94.9% yield).
Melting point onset = 160.00C (differential scanning calorimetry).
5 X-Ray Powder Diffraction The X-ray diffraction (XRD) patterns of crystalline solids are obtained on a Bruker D4 Endeavor X-ray powder diffractometer, equipped with a CuKa source A.
=
1.54060 A) and a Vantec detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40 in 20, with a step size of 0.009 in 20 and a scan rate of 0.5 10 seconds/step, and with 0.6 mm divergence, 5.28 fixed anti-scatter, and 9.5 mm detector slits. The dry powder is packed on a quartz sample holder and a smooth surface is obtained using a glass slide. The crystal form diffraction patterns are collected at ambient temperature and relative humidity. It is well known in the crystallography art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology or habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. Furthermore, it is also well known in the crystallography art that for any given crystal form the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature or humidity at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, a peak position variability of 0.2 in 20 will take into account these potential variations without hindering the unequivocal identification of the indicated crystal form. Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in units of 20), typically the more prominent peaks. (United States Pharmacopeia #35, National Formulary #30, Chapter <941>, pages 427-432, 2012). The crystal form diffraction patterns, collected at ambient temperature and relative humidity, are adjusted based on NBS standard reference material 675 (mica) with peaks at 8.853 and 26.774 degrees 2-theta.
A prepared sample of the co-crystal of Reference Compound 2 is characterized by an X-ray diffraction pattern using CuKa radiation as having diffraction peaks (2-theta

-33-values) as described in Table 3 below, and in particular having peaks at 18.2 in combination with one or more peaks selected from the group consisting of 16.0,25.4 , and 7.0 ; with a tolerance for the diffraction angles of 0.2 degrees.
Table 3 Reference Compound 2 I Angle ( 2-Theta) Relative Intensity Peak +1- 0.2 (% of most intense peak) 1 7.0 74.00 2 15.0 53.70 3 16.0 87.60 4 17.4 66.20 18.2 100.00 6 19.7 63.00 7 20.2 54.80 8 21.0 63.60 9 23.4 29.40 25.4 74.20 Alternative cocrystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone and 4-hydroxybenzoic acid.
To a 20 mL vial is added 2-(2,6-dichloropheny1)-141S,3R)-3-(hydroxymethyl)-10 5-(3-hydroxy-3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(111)-yflethanone (2.00 g, 1.00 equiv; 4.44 rrunoles). Acetone (4 mL) is added while stirring at room temperature. A clear solution is formed. 4-Hydroxybenzoic acid (0.756 g; 1.23 equiv;
5.47 mmoles) is added while stirring at room temperature. A slight suspension is formed then a thick suspension. The mixture is heated on hot plate to 60 C. Acetone in 1 mL
aliquots is added until a nice mixing suspension is observed at 60 C. Total acetone added is 9.00 mL (122.43 mmoles, 7.11 g). The temperature is held at -600C for several hours.
The mixture is cooled to room temperature and placed in refrigerator to improve

-34-recovery. The resulting solid is collected by vacuum filtration, rinsed with 2 mL of acetone and dried in a vacuum oven overnight at 400C to provide the title composition as a white crystalline solid. HPLC analysis demonstrates the molar ratio of 242,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1./)-yliethanone to 4-hydroxybenzoic acid in the cocrystal is one to one.
HPLC Analysis Column: Agilent ZORBAX Bonus-RP, Rapid Resolution, 4.6x75mm, 3.511 Column temperature: 30 C
Injection volume: 2 L
Detection: UV
2-(2,6-dichloropheny1)-14(1 S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(1H)-yl]ethanone (Reference Compound 1) @ 219nm 4-hydroxybenzoic acid @ 256nm Flow rate: 1.5 mi./min.
Mobile phase: A) 0.1% TFA in water B) 0.1% TFA in acetonitrile Gradient Table Time, minutes %A %13 9.5 23 77 12.1 23 77 13.0 5 95 16.0 5 95 16.1 95 5 20.0 95 5

-35-Relative Ratio Theoretical Molecular Compound Potency for 1 Assayed Potency (n=3) Weight to 1 Ref. Compound 450.4 76.5 % 81.06% + 0.15%

4-hydroxy-138.1 23.5% 23.74% + 0.20%
benzoic acid Second alternative cocrystallization of 2-(2,6-dichlorophenyI)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yljethanone and 4-hydroxybenzoic acid.
Compounds 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone (45.06 g, 0.1 mol) and 4-hydroxybenzoic acid (14.5 g, 1.05 mol eq) are slurried at 23 *C in 53:47 isopropyl alcohol:heptane (236 mL, 4 volumes) and heated to 65 C. The resulting solution is seeded with cocystals of 2-(2,6-dichloropherty1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone and 4-hydroxybenzoic acid (553 mg, 1.0 wt. % seed load) and stirred at 65 C for 30 minutes.
Heptane (943 mL, 16 volumes3) is added at 65 C over 4.6 hours. The slurry is stirred at 65 C for a further 30 minutes, cooled to 23 C over 2 hours, stirred overnight at 23 C, and vacuum filtered. The product solids are rinsed with 10:90 isopropyl alcohol:heptane (2 x 50 mL) and heptane (50 mL) then dried in a vacuum oven at 40 C for 2 hours to yield the title composition as a white crystalline product (51.3 g, 86.3 wt %
yield).
Melting point onset = 162.2 0C (differential scanning calorimetry).

-36-Example 1 Crystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone.
OH
OH
CI
0CI lei Approximately lOg of cocrystal 242,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yliethanone and 4-hydroxybenzoic acid is taken in a 500mL beaker and 100mL of deionized water added at room temperature. A solution of 50mL of 1N NaOH is slowly added. A suspension is formed. 100mL of chloroform is added gradually. The solids dissolved and a bi layer solution is formed. The organic layer is separated using a separatory funnel. The organic layer is evaporated at room temperature. To the resulting gummy material is added 20mL of heptane and stirred. The resulting solid is isolated under vacuum and air dried at room temperature in open dish to provide amorphous 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone. ). MS (m/z): 450.2(M+1 (35C1)).
Amorphous 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-543-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yl]ethanone (251.8mg) is taken in a 20m1, vial and 4mL of toluene is added. The resulting suspension is stirred overnight at room temperature. The crystalline solid is filtered under vacuum and air dried. Melting point onset ¨ 146.3 0C (differential scanning calorimetry).
Alternative crystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone.
Approximately 3g of amorphous 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone is taken in a 20mL vial and 10mL of acetone is added. A thick precipitate is

-37-formed immediately upon stirring at room temperature. Another 5m1., of acetone is added to dilute the slurry and stirred at room temperature for overnight. The resulting crystalline solid is isolated under vacuum and air dried.
X-Ray Powder Diffraction X-ray powder diffraction (XRPD) pattern is collected on a Bniker D8 Advance X-ray powder diffractometer equipped with a CuKa source (X=1.54056 A) and a Linxeye detector, and operating at 40 kV and 40 mA, with a 0.2 mm divergence slit.
Sample is scanned from 2 to 40 in 0.02 20 steps at a rate of 0.2 seconds per step. The crystal form diffraction patterns are collected at ambient temperature and relative humidity and peak position is adjusted based on NBS standard reference material 675 (mica) with peaks at 8.853 and 26.774 degrees 2-theta. It is well known in the crystallography art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology and habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. Furthermore, it is also well known in the crystallography art that for any given crystal form the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature or humidity at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, a peak position variability of 0.2 in 20 will take into account these potential variations without hindering the unequivocal identification of the indicated crystal form. Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in units of 20), typically the more prominent peaks. (United States Pharmacopeia #35, National Formulary #30, Chapter <941>, pages 427-432, 2012).
A prepared sample of the crystal of Example 1 is characterized by an X-ray diffraction pattern using CuKa radiation as having diffraction peaks (2-theta values) as described in Table 4 below, and, in particular, by having peaks at 9.150 in combination with one or more of the peaks selected from the group consisting of 19.4 , 16.1 , and 16.6 ; with a tolerance for the diffraction angles of 0.2 degrees.

-38-Table 4 Example 1 Peak Positions Relative Angle ( 20) Intensity (% of Peakd value (A) 0.2' most intense peak) 1 9.15 100.0 9.65 2 11.8 16.1 7.51 3 12.5 18.0 7.06 4 14.6 10.6 6.06 15.6 18.7 5.67 6 16.1 27.7 5.52 7 16.6 24.5 5.33 8 17.4 16.2 5.09 9 19.4 63.2 4.57 20.2 21.7 4.40 11 21.0 15.2 4.23 12 23.0 23.0 3.86 13 23.5 11.0 3.78 14 23.9 11.6 3.72 25.2 22.5 3.54

Claims (6)

We claim:

1. A crystalline form of 2-(2,6-dichlorophenyl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone.

2. The crystalline form of 2-(2,6-dichlorophenyl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone according to claim 1 characterized by an X-ray powder diffraction pattern using CuK.alpha. radiation having a diffraction peak at diffraction angle 2-theta of 9.15° in combination with one or more peaks selected from the group consisting of 19.4°, 16.1°, and 16.6°; with a tolerance for the diffraction angles of 0.2 degrees.

3. A pharmaceutical composition comprising a crystalline compound according to claim 1 or 2, and a pharmaceutically acceptable carrier, diluent or excipient.

4. A method of treating Parkinson's disease comprising administrating to a patient in need thereof an effective amount of a crystalline compound of claim 1 or 2.

5. A crystalline compound as claimed in claim 1 or 2 for use in therapy.

6. A crystalline compound as claimed in claim 1 or 2 for use in the treatment of Parkinson's disease.